Insulin-dependent diabetes mellitus (IDDM) (also known as type 1 diabetes) primarily afflicts young people. Although insulin is available for treatment, the several-fold increased morbidity and mortality associated with this disease require the development of early diagnostic and preventive methods. The destruction of pancreatic .beta.-cells (which are the insulin-secreting cells of the islets of Langerhans) that precedes the clinical onset of IDDM, is mediated by autoimmune mechanisms. Among the most thoroughly studied autoimmune abnormalities associated with the disease is the high incidence of circulating .beta.-cell specific autoantibodies at the time of diagnosis. Family studies have shown that the autoantibodies appear prior to overt IDDM by a number of years, suggesting a long prodromal period of humoral autoimmunity before clinical symptoms emerge. The family studies have also documented a slow, progressive loss of insulin response to intravenous glucose in the years preceding diagnosis. The presence of .beta.-cell specific autoantibodies in the prediabetic period is likely to reflect the ongoihg autoimmune process, one that eventually leads to critical .beta.-cell depletion and insulin dependency. It has been estimated that only 10% of the total .beta.-cell mass remains at the time of clinical onset.
The target of autoantibodies in pancreatic .beta.-cells in IDDM was originally identified as a 64 kDa autoantigen by immunoprecipitation experiments using detergent lysates of human islets (Baekkeskov et al. (1982), Nature 298:167-169). Antibodies to the 64 kDa autoantigen precede the clinical onset of IDDM and have been shown to have an incidence of about 80% at clinical onset and during the prediabetic period (Baekkeskov et al. (1987), J. Clin. Invest. 79:926-934; Atkinson et al. (1990), Lancet 335:1357-1360; and Christie et al. (1988), Diabetologia 31:597-602) (each of which is incorporated by reference in its entirety for all purposes). The rat and human 64 kDa protein are highly homologous with regard to autoantigenic epitopes (Christie et al. (1990), J. Boil. Chem. 265:376-381) (incorporated by reference in its entirety for all purposes). The 64 kDa autoantigen in islets of Langerhans is detected in three different forms with regard to hydrophobicity and compartmentalization: a hydrophilic soluble form of 65 kDa and Pi of approximately 7.1; a 64 kDa hydrophobic form, which is soluble or of a low membrane avidity and has a Pi of approximately 6.7; and a hydrophobic firmly membrane anchored form of the same electrophoretic mobility and Pi. Both the membrane bound and the soluble hydrophobic 64 kDa forms exist as two isoforms, .alpha. and .beta. which have identical Pi and hydrophobic properties but differ by approximately 1 kDa (Baekkeskov et al. (1989), Diabetes 38:1133-1141) (incorporated by reference in its entirety for all purposes). The 64 kDa autoantigen was found to be .beta.-cell specific in an analysis of a number of tissues, which did not include the brain (Christie et al., supra).
It has recently been shown that the 64 kDa autoantigen of pancreatic .beta.-cells is glutamic acid decarboxylase (GAD, L-glutamate 1-carboxy-lyase, EC 4.1.1.15). The GAD enzyme synthesizes GABA from glutamic acid and is an abundant protein of GABA-secreting neurons in the central nervous system (CNS). See copending application, Ser. No. 07/756,207; Baekkeskov et al. (1990), Nature 347:151-157 (incorporated by reference in its entirety for all purposes).
GAD is an abundant and partially-characterized protein of GABA-secreting neurons in the central nervous system. The GAD enzyme has two forms encoded by two distinct non-allelic genes, GAD.sub.67 and GAD.sub.65 (also known as GAD-1 and GAD-2), which may have developed from a common ancestral gene during vertebrate phylogeny. GAD.sub.67 and GAD.sub.65 are highly diverse in the first 95 amino acids but share significant (approx. 75%) homology in the rest of the molecule. Both have a proteolytic hot spot 80-90 amino acids from the N-terminus (Christgau et al. (1991), J. Boil. Chem. 266:21257-21264; Christgau et al. (1992), J. Cell Biol. 118:309-320) (incorporated by reference in their entirety for all purposes), which may represent a domain boundary. The N-terminal domain harbors the post-translational modifications which result in anchoring of GAD.sub.65 to the membrane of synaptic vesicles and control the distinct subcellular localization of this protein.
In brain tissue, both GAD.sub.65 and GAD.sub.67 are produced (Bu et al. (1992), Proc. Natl. Acad. Sci. USA 89:2115-2119; Kaufman et al. (1986), Science 232:1138-1140; Chang & Gottlieb (1988), J. Neurosci. 8:2123-2130) (each of which is incorporated by reference in its entirety for all purposes). Some species express both GAD proteins in their pancreatic islets. However, in human islets only GAD.sub.65 is expressed (Karlsen et al. (1991), Proc. Natl. Acad. Sci. (USA) 88:8337-8341; Karlsen et al. (1992), Diabetes 41:1355-1359) (incorporated by reference in their entirety for all purposes). Immunogenic crossreactivity between isolates of GAD.sub.65 and GAD.sub.67 from different vertebrate species indicates a high degree of conservation of antigenic determinants from rodents to humans (Legay et al. (1986), J. Neurochem. 46:1478-1486). Consistent with this observation, human GAD.sub.65 and GAD.sub.67 polypeptides share more than 90% amino-acid sequence identity with cognate polypeptides in other mammals. Bu et al., supra.
The cDNAs of human CNS GAD.sub.67 and GAD.sub.65 have been cloned and sequenced (Bu et al., supra). Karlsen et al. (1991), supra, have reported sequence data for human pancreatic beta cell GAD.sub.65. DNA sequence information is also available for rat CNS GAD.sub.65 and GAD.sub.67 (Erlander et al. (1991), Neuron 7:91-100; Julien et al. (1990), J. Neurochem. 54:703-705) and rat beta cell GAD.sub.65 (Michelson et al. (1991), Proc. Natl. Acad. Sci. (USA) 88:8754-8758) (each of which is incorporated by reference in its entirety for all purposes).
The demonstrated equivalence of the 64 kDa IDDM autoantigen and GAD explains earlier observations linking IDDM with a rare, but severe, neurological disease termed stiff man syndrome, in which GAD has been recognized as the predominant autoantigen (Solimena et al. (1988), N. Engl. J. Med. 318:1012-1020; Solimena et al. (1990), N. Engl. J. Med. 322:1555-1560) (incorporated by reference in their entirety for all purposes). Almost all the GABA-ergic neuron autoantibody positive patients were also positive for islet cell cytoplasmic antibodies, and one third had IDDM. In addition, autoantibodies to GABA-ergic neurons were detected in 3 of 74 IDDM patients without SMS (Solimena et al. (1988), supra, and Solimena et al. (1990), supra). Other studies have also reported a high incidence of IDDM in SMS patients (Lorish et al. (1989), Mayo Clin. Proc. 64:629-636).
The demonstrated equivalence of the 64 kDa antigen and GAD has also led to some improvement in methods of diagnosing IDDM. Previously, the 64 kDa autoantigen had only been identified in the pancreatic .beta.-cell, and could not be purified in sufficient quantities to allow cloning, sequencing or other characterization that would have permitted large-scale preparation of reagents necessary for efficient detection or therapy. By contrast, the abundance of GAD in brain allows facile production by cloning, or otherwise, of large amounts of GAD protein (either GAD.sub.65 or GAD.sub.67) as a reagent for diagnosis. See co-pending application Ser. No. 07/756,207.
Although an improvement on prior methods, diagnosis using full-length forms of purified GAD protein is still not entirely satisfactory. GAD molecules have a lipid modification in the N-terminal region and are therefore insoluble except in the presence of detergent. The insolubility of full-length GAD molecules hampers purification, and use of GAD, in simple assays like immunoprecipitation, ELISA or radioimmunoassay. Moreover, use of full-length GAD as a diagnostic reagent does not distinguish between different classes of GAD autoantibodies, which may be diagnostic of different temporal stages of an autoimmune disease and/or different diseases. Furthermore, insoluble GAD proteins are unsuitable for in vivo administration as therapeutic reagents.
Based on the foregoing, it is apparent that a need exists for improved reagents and methods for diagnosing and treating patients having, or at risk of, IDDM and stiff man syndrome. The present invention fulfills this and other needs.